CYTOTOXIC EXPOSURE OF GREEN ALGAS CHLAMYDOMONAS PETERFII GERLOFF IN RADON AEROSOLS*

D. CIORBA1, A. TRUTA 1
1

Environment Science and Engineering Faculty, Babes-Bolyai Faculty, Cluj-Napoca, Romania, E-mail: ciorbad@yahoo.com; E-mail: luciadinapopa@yahoo.com Received November 15, 2012

OECD, Guidelines for the Testing of Chemicals recommend the alga growth inhibition test, in ecotoxicological studies, for assesing the exposure. Regarding the Radon exposure as a toxic exposure, we try to observe the induced effects, as a consequence of alpha particle deposition, on our Chlamydomonas culture kept 10 minutes at 400C and relative humidity 70%, in our in vitro exposure system, when the Radon concentrations was 500 Bq/m3 and 1000 Bq/m3. Control sample follow the same exposure, but for a background Radon level of 48 Bq/m3. The decrease of surviving rate has been observed, especially for 1000 Bq/m3 scenarios. In this light, this test shoud be very good for study of environmental exposure into the thermal area. Key words: Chlamydomonas, surviving, Radon, aerosols, worst scenarios.

1. INTRODUCTION More than half of the natural radiation dose which affect the population is caused by radon and its progenies. A high priority to re-evaluation of risks needs new data about occupational and domestic exposure. From this point the unicellular green algae should be a good model for study the entry of alpha particle, bioaccumulation, binding and degradation, involving the process not normally encountered in toxicology, [1]. The green algae as biological systems are frequently used for testing of toxicity related to chemical safety and environmental health due to their facility to give a feedback in a very short time. In radiation, the green algae were studied especially for aquatic environment when has been observed that group of chlorophyta, has a great resistance to higher radiation doses, [2]. The response (survival) of green alga to Radon aerosols deposition is not known or less studied. Into the thermal area, Radon concentrations of thermal waters and in consequence, the aerosols radioactivity are changing in time, [3]. The indoor 222Rn,
Paper presented at the First East European Radon Symposium FERAS 2012, September 25, 2012, Cluj-Napoca, Romania. Rom. Journ. Phys., Vol. 58, Supplement, P. S73S83, Bucharest, 2013
*

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attached and unattached progenies, represent an agent of additional radiation burden, and the variations of aerosols depositions according with their size, distribution, ultrafine fraction, equilibrium fraction, and hygroscopicity should be interesting for estimate the short-term health impact, the treatment possibility or occupational exposure for bathers and working personnel, [4-7]. We try to reproduce as much as possible the thermal area conditions regarding Rn222 concentrations, temperature and humidity inside of our original irradiation system, named RADOSIV 2, [8]. Chlamydomonas Peterfii Gerloff, was the biological exposed model. These chlorophytas algae have a great similarity to Chlamydomonas Reinhardtii - the very well known genetic model, [9]. Our choice was studying the exposure at 500 Bq/m3 and 1000 Bq/m3, considering these concentrations as two of the worst scenarios, because according to Somlai et al., for workplaces within the EU, the action levels should be set in this range, [5]. Yearly average radon concentration in workplaces was 1000 Bq/m3 in air, [10]. These values are based on an assumed equilibrium factor of 0.4, (Radiation Protection 88), the action level that should not be exceeded for protection of category A workers (annual doses higher than 6 mSv), [11]. On the other hand, ICRP 103 since 2007, kept the radon upper values at 1500 Bq m-3 for workplaces [12]. We try to observe any abnormal appearance of the algal cells caused by the exposure to these known Radon concentrations. The cytotoxic endpoints followed were cells survival, mutant chlorophyll, changes of population distribution, with consequence on life cycle and yield production. MATERIAL AND METHODS Irradiation and survival assays Our in Vitro Exposure System has a cubic shape with sides of 50 cm, made of metal, except one wall of glass which allows observation of samples during the experiment. Each metal wall has an extern pocket, where can be introduced (optional) a radioactive source, for increasing the indoor doses. In this experiment the irradiation was made by keeping in the pocket the pitchblende ore. Radon gas inside was generated by this source. Inside of RADOSIV2, picture 1, was also an outlet for connecting an electric kitchen range. The different aerosol concentrations have been obtained by warming of water. Our samples - algal culture were kept in Petri dishes, with diameter of 10 cm. When the Radon aerosols concentrations were 500 Bq/m3 (the I-st worst scenario) and 1000 Bq/m3 (the II-nd worst scenario), we introduced the Chlamydomonas culture, for 10 minutes, at 400C and relative humidity of 70% . Control sample follow the same exposure, but for a background Radon level of 48 Bq/m3. The indoor doses were monitored by means of a Lucas Cell Radon Scout, Radim 2P, Radim 3A, Rad 7 and Sarad Radon detector. Chlamydomonas Peterfii Gerloff, strain AICB 528 was provided from Algal Culture Collections of Biology Faculty, Babes-Bolyai University. We

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analyzing the algal culture, respecting the OECD guidelines 2011, regarding exposure hazard assessment, for 3 days, post irradiation, [13]. Manual cells counting using Axioplan, Zeiss fluorescence microscope at 250X total magnification was made. Also, the cells density was assessed using a hemocytometer counting chamber, combined with an UV visible spectrophotometer, (UV mini 1240, Shimadzu), based on calibration curve at 600 nm. Blue Evans Dye has been used for study of population distributions based on cells viability, [14].

Picture 1 In Vitro Irradiation System, RADOSIV 2.

We classified the cells viability according to colors penetration into the cell. So, we considered CM, the cell death with blue coloration of all body; CAP, the cell which going into apoptosis process, with blue coloration only of 50% from cells body; CAM, the viable cells, having coloration only at the outside membranes and CV, the viable cell, without penetration of color inside of body, picture 2.

Picture 2 Viability Scale of unicell chlamydomonas. Legend: CM = cell death; CAP = apoptotic cell; CAM = mature cell; CV = young cell.

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Chllorophyll measurement Media: 80% (v/v) acetone, in water. 10 l of the extract has been analyzed to 1 ml 80% (v/v) acetone in a 1-ml Eppendorf tube. The next step was vortex and incubation for 15 min on ice and in the dark, followed by centrifugation for 15 min at 16,000 g and absorbance measuring at 600 nm, procedure adapted from Arnon [15], and presented at EMBO Course from 2006, [16]. Data analysis The experiments were made in copy (double test) from independently grown algal cultures. Data points in figures mean average per 10 separate microscopic fields. Error bars represent standard deviation. The survival fractions for some kind of cells were obtained by Smoothing FFT of Data Points post irradiation at 24 hour, 48 hours and 72 hours. 2. RESULTS The growth inhibition rate - Screening of cells survivors show us an inhibition of growth rate post irradiation. If for the control, we have an increase growth rate of 0.955/day, for the first scenarios 500 Bq/m3, we observe a non significant increase, the rate being 0.062/day post irradiation. For the second scenarios, 1000 Bq/m3, we observe a decrease of Chlamydomonas survivors, the decrease of growth rate was 0.399/day, Figure 1.
Control 500Bqm3 1000Bqm3

Cells number/ microscopic cadru

200 180 160 140 120 100 80 60 0 1 2 3

Time, days
Fig. 1 The grow inhibition rate of Chlamydomonas Peterfii Gerloff post irradiation with 500 Bq/m3 and 1000 Bq/m3, Radon aerosols. Standard deviation is represented for each point. Data points in figures mean average per 10 separate microscopic fields.

The linear equations of cells growth and the process rate (exponential analysis) are presented in Table 1.

Cytotoxic exposure of green algas chlamydomonas Peterfii Gerloff Table 1 The linear equation of cells growth, for green algas Chlamydomonas Peterfii Gerloff, in the next three days, after the irradiation with Radon aerosols Linear ecuation of cell's growth non irradiated Control y = 31.62 x + 31.44 growth/ inhibition rate R2 = 0.955 R2 = 0.062 R2 = 0.399 Remarks exponential increase non significant increase decrease

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irradiated 500Bq/m3 y = 0.486 x + 63.31 irradiated 1000Bq/m3 y = 4.03 x + 87.25

Analysis of cells distribution according to the viability scale, (picture 2), show us that the biggest number of individuals, in population range is the chlamydomonas cells who fix the coloration only at membrane outside (CAM population according with our mentions), Figure 2a. At the same time we observed the significant decrease of CAM populations both for 500 Bq/m3 and 1000 Bq/m3 (Figure 2b, 2c and Figure 3).
Cell's Distribution in Control Cell's number/microscopic cadru
120 100 80 60 40 20 0
CV CM CAM CAP

Cell's number/microscopic cadru

Cell's Distribution Cell's number/microscopic cadru

60 50 40 30 20 10 0 1 2

in Sample exposed at 500 Bq/m3

CV CM CAM CAP

Cell's Distribution
120 100 80 60 40 20 0

in Sample exposed at 1000 Bq/m3

CV CM CAM CAP

Time, days

Time, days

Time, days

a)

b)

c)

Fig. 2 Analysing the Chlamydomonas Peterfii Gerloffs viability, according to the Blue Evans dye penetration into the cells post irradiation with Radon aerosols. Standard deviation is represented for each point. Data points in figures mean average per 10 separate microscopic fields, when CM = cell death; CAP = apoptotic cell; CAM = mature cell; CV = young cell Legend: a) Control; b) 500 Bq/m3; c) 1000 Bq/m3.

We consider that overall observed inhibition of algal growth is a consequence of decrease in surviving rate, especially for CAM population, Figure 3. The apparent lower surviving rate of Chlamydomonas in the II-nd scenarios (1000 Bq/m3) compared with the I-st scenarios (500 Bq/m3) for CAM populations, should be caused by the increase rate of necrosis/ apoptosis process, for CM and CAP populations, Figure 4.

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% of Surviving, CAM Cells

1,0 0,9 0,8 0,7 0,6 0,5 0,4 0,3 0,0 0,5 1,0 1,5 2,0 2,5 3,0 3,5

1000 Bq/m3

500 Bq/m3
4,0

Days Post Irradiation

Fig. 3 Surviving rate of CAM population, at the 500 Bq/m3 and 1000 Bq/m3, Radon aerosols. The lines mean the Smoothing FFT of Data Points.
0,36

% of (CAP+CM Population)

0,32 0,28 0,24 0,20 0,16 0,12 0,08 0,04 1,0 1,5 2,0 2,5 3,0 3,5 4,0

1000 Bq/m3

500 Bq/m3

Days Post Irradiation

Fig. 4 The increase of apoptosis/necrosis process (CM and CAP populations) post irradiation with 500 Bq/m3 and 1000 Bq/m3, Radon aerosols. The lines mean the Smoothing FFT of Data Points.

ChlorophyllAbsorbance and Spectrum The sample absorbance in vivo, provide information related to the photosynthetic chain function by understanding of various electron transfer within different complexes and thereby to characterize their function. An absorption changes is equivalent with change of the extinction coefficient. The ratio between the variation in light intensity and the intensity transmitted before the exciting is thus proportional to the absorption change. The signal being proportional with sample concentration, the former can be easily increased by increasing cells number latter. Yet, increasing of cells density (concentration), obviously results the increasing of sample overall absorption. In Figure 5, the increased absorbance at 1000 Bq/m3 should be explained only through reverse of intracellular content according with necrosis/apoptosis process presented before, which in analytical terms is equivalent with noise of measurement.

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0,060 0,055 0,050 0,045 0,040 0,035 0,030 0,025 0,020 0,015 0,010 0,005 0,000 2,0 2,5 3,0 3,5 4,0 4,5 5,0 5,5

AU, 600 nm

6,0

Time, days
Fig. 5 Absorption of chlamydomonas culture (in vivo), at 600 nm for control and post irradiation with 500 Bq/m3 and 1000 Bq/m3. Standard deviation is also, represented for each data point.

The chlorophyll content and chlorophyll quality has been significantly changed post irradiation. As a consequence of alpha particles deposition on the plates, different absorbances peak has been registered, Table 2.
Table 2 The absorbances peak (nm), in the 3-th and the 4-th day post irradiation Control the 3-th day 667 nm the 4-th day 667.5 nm 617 nm 434 nm 435 nm 457 nm 433 nm 467 nm 435 nm 500 Bq/m3 the 3-th day 664 nm the 4-th day 667.5 nm 1000 Bq/m3 the 3-th day 659 nm 741 nm 774.5 nm 432 nm 424 nm the 4-th day 667.5 nm

DISCUSSIONS The algal growth inhibition test is a more dynamic test system than most other short-term aquatic toxicity tests, which are used generally for checking a substance or the decline concentration of a test substance, [17]. The assessment of exposure hazard using Chlamydomonas models gives us the possibility to follow in vivo the influence of low doses and estimate the risk. Experiments conducted in human and other mammalian cells grown in tissue

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culture exposed to low doses can provide some information for indicating the mechanisms that may be involved, but only through in vitro cellular experiments or using animals which have some inconvenience: higher number of animals, different time of exposure; higher uncertainty in assessing of early life exposure risk, higher costs, etc. Plant survival and other eukaryotic cells, is essential for an organism. Based on Envirhom Raport, particularly to green algae Chlamydomonas Reinhardti, has been observed the decrease of surviving rate for bioacumulated depleted 233 Uranium, when EC 50 concentrations was 131 nM after 24 hours, 151 nM after 48 hours and 231 nM after 72 hours, [18], [19]. Rn is one product in the radioactive decay series of 238U. The deposition of radon aerosols and then attached radon decay products on the plates depends strongly to the local distribution of aerosol-particles present in the indoor air. Thus, the toxic effects registered must be in accord with the plate surfaces, with cells number, with Chlamydomonas Peterfii Gerloff organismsensitivity or individual sensitivity. The life cycle of C. Reinhardti is easily changed in stress conditions by means of sexual cycle, [20]. The exposure to different Radon aerosols concentrations induce a genotoxic stress with consequence of chlaydomonas life cycle, observed like decreasing of cells surviving rate and alterations of individual distribution, with effect of yield production. As a result, we observe a highly decrease of vegetative cells, CAM population and increasing the necrotic/apoptotic cells, CM and CAP population, function of age sensitivities. The unicell Chlamydomonas adjust their rate of photosynthesis according to the environmental conditions. Plants and green algae are particularly susceptible to DNA damage especially that caused by UV light, due to their light dependency for photosynthesis, [21], [22]. As we know the energetically alpha particle emitted to Radon and its progenitors induce oxidative damage with appearance of reactive oxygen species (ROS) in life systems. Due to increased absorbance observed post irradiation with 1000 Bq/m3, we consider that such events have happened, with effect on the cellular components and photosynthetic apparatus. The action of alpha particle upon the chloroplast should be similarly to Goldschmidt-Clermont suggestion, related to the direct influence on mutagenesis, [23]. In their opinion, the mutants with defects are generally photosynthetic deficient, light sensitive, and can be identified by their high chlorophyll fluorescence. At the same time, the mutants with defects, have different viability, [24]. In our situation this aspect is relevant through different rate of apoptosis/necrosis process. Possibly, due to mutagenesis, we registered many peaks in chlorophyll absorbance post irradiation. The two peck of absorbance higher than 700 nm was registered only after the irradiation with 1000 Bq/m3, which suggests the induction of photoinhibition through effect upon PSII system, knowing that dissipation of energy excess by fluorescence occurs mostly at PSII.

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Based on these results, the unicell chlamydomonas is a versatile system for the study of a variety of molecular and cellular processes in radiation, being the most closely cell plant model with others, animal cells. In radiation protection, in the same way, for unicell chlamydomonas, animals and humans, is possible that some individuals to be more radiation-sensitive than others, for genetic reasons. So, the analysis between the two different worst case scenarios, give us the possibility to analyse how the low doses produce different and potentially more harmful effects in sensitive individuals, [25]. Another aspect, relevant in this field, will be study of pattern deposition related with algal inhibition growth rate and the bystander effect to different low doses. The penetration of alpha particle into the chlamydomonas cells after the radon aerosols deposition and the disintegration time of short-lived radon daughters should be interesting to be analysed versus the human cell line (epithelial or skin fibroblast) with develops the model penetration through the skin, [26]. CONCLUSION The analyze of radiation exposure from environment needs new testing methods, with low complexity level, short time and reduced costs, compared with actual methods, focused especially on vertebrate organisms answer. Unicells Chlamydomonas represent a very good in vivo model, possible to be followed comparative with other in vitro model, animal or human, for assessing the exposure to different indoor Radon aerosols concentrations. The shorter surviving rate of cells, with consequence of populations distribution was observed, according to the irradiation dose. The exposure induced oxidative damage through reactive oxygen species with effects on the cellular components and photosynthetic apparatus. The most important inhibition of growth alga, 0.399 per day, has been registered at 1000 Bq/m3, which represents a really worst case scenario, according to our results, raising the question related to the occupational safety in area with variable Radon aerosols deposition.
Acknowledgements. The authors would like to thanks to Professor Dr. Constantin Cosma, who provides the pechblenda radioactive ore used for Radon detectors calibration and also, to Professor Dr. Nicolae Dragos for providing the Chlamydomonas Peterfii Gerloff culture, and also to POSDRU/89/1.5/S/60 189 for financial support.

REFERENCES
1. BEIR VI, Health Effects of Exposure to Radon: Time for Reassessment? ISBN: 978-0-309-050876, http://books.nap.edu/catalog/4777.html, (1999). 2. C. Mothershill, C. Seymour, Radiation induced bystander effects: past history and future directions Radiat.Res. 155, 759767 (2001).

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D. Ciorba, A. Truta

10

3. F. Steinhausler, Radon spas: source term, doses and risk assessment., Radiat. Prot. Dosim. 24, 257259, (1998). 4. N. Janoki, J. Somlai, V. Jobbagy, T. Kovacs, Radiological Survey of Thermal Bath in Igal Hungary, Studia Universitas Babes-Bolyai, Ambientum, 1, 5561, (2009). 5. J. Somlai, A. Torma, P. Dombovri, N. Kvsi, K. Nagy, T. Kovcs, Contribution of 222Rn, 226Ra, 234U and 238U radionuclides to the occupational and patient exposure in Heviz-Spas in Hungary. J. Radioanal. Nucl. Chem., 272 (1), 101106, (2007). 6. T. Kovacs, N. Kavasi, C. Nemeth, J. Somlai, T. Vigh, G. Szeiler, Parallel Radon and Thoron Survey at Special Underground Circumstances, Studia Universitas Babes-Bolyai, Ambientum, 1, 6369, (2009). 7 E. Vogiannis, D. Nikolopoulos, A. Louizi, C.P. Halvadakis, Radon Exposure in the Thermal Spas of Lesvos Island Greece, Radiation Protection Dosimetry, 111(1): 121127, doi:10.1093/rpd/nch373, (2004). 8. D. Ciorba, M. Moldovan, Radon System of Irradiation In Vitro RADOSIV 2, Ambientum, vol III, ISSN 1843-3855, accepted for publication in 2011. 9. G.S. Chankova, J. A. Matos, F. Simoes, P. E. Bryant, Adaptive response of a new radioresistant strain of Chlamydomonas Reinhardtii and correlation with increased DNA double-strand break rejoining, Int. J. Radiat. Biol., 81(7), 509514, (2005). 10. International Basic Safety Standards for Protection against Ionizing Radiation and for the Safety of Radiation Sources, IAEA Safety Series No. 115, (1996). 11. Radiation Protection 88, Recommendations for the implementation of Title VII of the European Basic Safety Standards concerning significant increase due to natural radiation sources, (1997). 12. ICRP Publication 103. The 2007 Recommendations of the International Commission on Radiological Protection. Ann. ICRP 37, 24, (2007). 13. Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures. Environmental Health and Safety Publications. Series on Testing and Assessment, no. 23. Organisation for Economic Co-operation and Development, Paris, (2011). 14. P. Brian, A. Piasecki, R. Kenneth, B. Diller, J. Jerry Brand, Cryopreservation of Chlamydomonas Reinhardtii: A cause of low viability at high cell density, Cryobiology 58: 103109, (2009). 15. D. L. Arnon, Copper enzymes in isolated chloroplasts. Polyphenoloxidase in Beta vulgaris. Plant Physiol. 24, 115, (1949). 16. EMBO Practical Course, Molecular Genetics of Chlamydomonas, Laboratory Protocols, Geneva, (2006). 17. International Standard ISO 14442, International Organization for Standardization Water quality Guidance for algal growth inhibition tests with poorly soluble materials, volatile compounds, metals and waste water, (1998). 18. Report DRPH 2005-07 et DEI 2005-05, Envirom, Bioaccumulation of Radionuclides in situations of chronic exposure of ecosystems and members of the public, Direction de LEnvironment et de LIntervention, IRSN, (2005). 19. N.A. Beresford, M. Balonov, K. Beaugelin-Seiller, J. Brown, D. Copplestone, J.L. Hingston, J. Horyna, A. Hosseini, B.J. Howard, S. Kamboj, T. Nedveckaite, G. Olyslaegers, T. Sazykina, I. Vives, J. Batlle, T.L. Yankovich, Yu C An international comparison of models and approaches for the estimation of the radiological exposure of non-human biota. Appl Radiat Isot. doi: 10.1016/j.apradiso.2008.04.009, (2008). 20. U. Goodenough, H. Lin, J. H. Lee, Sex determination in Chlamydomonas, Seminars in Cell & Developmental Biology 18, 35036, (2007). 21. J. D. Rochaix, Assembly, Function, and Dynamics of the Photosynthetic Machinery in Chlamydomonas Reinhardtii, Plant Physiol., 127, www.plantphysiol.org/cgi/doi/10. 1104/ pp.010628, (2001).

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22. A. Sirikhachornkit, J.W. Shin, I. Baroli, K. Niyogi, Replacement of alpha-Tocopherol by betaTocopherol Enhances Resistance to Photooxidative Stress in a Xanthophyll-Deficient Strain of Chlamydomonas Reinhardtii, Eukaryotic Cell, 8(11), 16481657, (2009). 23. M. Goldschmidt-Clermont, Coordination of nuclear and chloroplast gene expression in plant cells, Int Rev Cytol 177:115180, (1998). 24. K. Kindle, High-frequency nuclear transformation of Chlamydomonas reinhardtii. Proc. Natl. Acad. USA 87: 12281232, (1990). 25. R. E. J. Mitchel, Low Doses of Radiation are protective in vitro and in vivo: Evolutionary Origins, Dose-Response 4(2), 7590, (2006). 26. H. Lettner, A. Hubmer and W. Hofmann, Rn progeny deposition on the skin and in the lung: what is more important in radon therapy? Book of Abstracts, 12th International Congress of the Internat. Radiation Protection Association (IRPA 12), Buenos Aires, Argentina, 473, (2008).